B. Mathods
7.2 Current neuronal differentiation
P19 cells were plated in tissue culture dish at a density of 2 x 105 cells/ml in 10% fetal bovine serum (FBS)/DMEM. To induce neuronal differentiation, the media were removed, and the cells were washed with HBSS and was replaced with N2 serum-free medium with or without final concentration 10 µM forskolin. The cells maintained for 4 days. The same protocol was used for P19/hShhN cells.
- Conventional method
- Current method
Neuronal cells P19 cells Embryoid body
10% FBS N2
4-8d
ShhN, N2
P19 cells Neuronal cells
4d RA +2d/-2d
Fig. 4. The method for neural induction of P19 cells.
8. Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)
Total RNA was isolated using a RNAzolTM B and cDNA was synthesized using First-strand cDNA synthesis kit and 1㎍ of total RNA following the manufacture’s instructions. The PCR reactions were carried out according to standard protocols. Primer sequences (forward and reverse) and product sizes (base pairs) were as follows (Table 1). The PCR products were analyzed on a 1.5% agarose gel.
Table 1. Primer sequence for RT-PCR
Forward Reverse Size (bp)
9. Western blotting
Whole cell lysates were prepared as follows; Cells were washed with cold phosphate buffered saline (PBS) twice and drained. Cells were resuspended in RIPA buffer [150mM Sodium chloride, 1% NP-40, 0.1% SDS, 50mM Tris-HCl (pH8.0), 0.5% sodium deoxycholate] and kept on ice for 5 min. After centrifugation at 12000 rpm for 20min, the supernatant was collected and protein content was assayed by Bradford method. Each 60 ㎍ proteins were separated on 8% or 12% SDS polyacrylamide gel electrophoresis and transferred to PVDF membranes. Membranes were incubated in blocking solution [5% nonfat dry milk in 10mM Tris-HCl (ph7.4), 100mM NaCl, and 0.1% Tween 20] for 1 hour at room temperature and with mouse anti- ShhN (1:100), mouse anti-βIII -tubulin (Tuj-1, 1:1,000), mouse anti-neuronal cell adhesion molecule (NCAM, 1:1,000), mouse anti-neurofilament-160kD (NF-M, 1:1,000), mouse anti-glial fibrillary acidic protein (GFAP, 1:1,000), and mouse anti-2', 3'-cyclic nucleotide 3'-phosphodiesterase (CNPase, 1:1,000) antibody in TTBS [10mM Tris-HCl (ph7.4), 100mM NaCl, and 0.1% Tween 20] solution for overnight at 4°C. The secondary antibody was horseradish-peroxidase (HRP)-conjugated to goat anti-mouse IgG (1:5,000) or anti-goat IgG (1:5000) and the proteins were visualized using an enhanced chemiluminescence (ECL) kit following the manufacture’s recommendation.
10. Immunocytochemistry
Differentiated P19 and P19/hShhN cells were fixed with 2~4%
paraformaldehyde solution or 100% methanol for 10 min at 4℃. The fixed was washed with PBS solution, three times and incubated with 10% normal horse serum and goat serum, 0.1% BSA and 0.03% Triton X-100 in PBS (PBS-T) for 1hour. After sufficient washing, the cells were incubated with primary antibodies overnight at 4℃.
The following primary antibodies were used at the concentrations given: Rat 401 (1:200), GFP (1:200), Ki-67 (1:200), Tuj1 (1:200), NFM (1:200), TH (1:200), Isl-1 (1:200), ChAT (1:200) and Hb9 (1:200). Following several washing with 0.03%
Triton X-100 containing PBS-T solution, cells were incubated in FITC conjugated anti-mouse IgG (1:500), anti-rabbit IgG (1:500), Alexa 488 anti-mouse IgG (1:500), or Alexa 594 anti-rabbit IgG (1:500) secondary antibody for 1 hour in dark chamber.
After sufficient washing with PBS-T, cells were mounted on acras or cover slips by using Vectashield (mounting medium for fluorescence, Vector) with DAPI or Hoechst (Molecular probe) and photographed using a fluorescent microscope.
III. RESULTS
1. Expression of hShhN in P19 cells
To determine whether Shh could play a role in neuronal differentiation, the expression of components of the Shh signaling pathway in P19 cells was analyzed in western blot. Shh was expressed in P19 cells (Figure 5 A). The N-terminal fragment receives two lipid modifications and, after being secreted, can tether to the membrane of producing cells or diffuse, establishing concentration gradients (Porter et al. 1996; Pepinsky et al. 1998; Lewis et al. 2001; Zeng et al. 2001). Medium conditioned by P19/hShhN cells contained Shh-N (Figure 5 A). The components of the Shh receptor complex, Ptc and Smo, as well as the three Gli transcription factors involved downstream of Shh signaling, have been demonstrated by RT-PCR to be expressed on P19 cells (Figure 5 B).
A.
P19 P19/hShhN
C M C M
Shh
B.
Smo
Ptc
Gli
GAPDH P19
P19/hShhN
Fig. 5. Generation of ShhN-Producing P19 Cell Lines. (A) Cells, P19 and P19/hShhN, were harvested and media collected, separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to PVDF membrane, and probed with antibodies direct against the anti-ShhN. ShhN protein as a 20 kDa was detected both cell extracts and medium in P19/hShhN cell. (B) Expression level of Smo, Ptc and Gli mRNA was measured by RT-PCR.
2. Preparation of 5E1, a neutralizing antibody against Shh
5E1 hybridoma cells were obtained from DSHB (IA, U.S.A). The cells were grown in the presence of HAT (Hypoxanthine-Aminopterin Thymidine). The cells were injected into the peritoneal cavity of BalbC female (6~8 week) to obtain ascitic fluid. The titer of 5E1 ascitic fluid was determined by ELISA using P19/hShhN conditioned medium as an antigen. 5E1 reacted with secreted ShhN in ELISA at dilutions up to >1:1000 (ascitic fluid) (Fig 6 A). IgG fractions were obtained by affinity purification on protein G–sepharose (Fig 6 B). Purified 5E1 were measured the concentration of ShhN by coomassie blue staining and showed neutralizing activity with ShhN in growth kinetics. P19/hShhN were inhibited proliferation at 10~30 ㎍/ml 5E1 (Fig 7).
3. The Concentration of secreted Shh
Shh detected in media conditioned by P19/hShhN using an ELISA. As this concentration of Shh is near the low limit of detection we used the same ELISA protocol to measure Shh in growth media containing 10% FBS, conditioned media form P19/hShhN and differentiation media containing N2 supplement in DMEM:F12 from P19/hShh. The concentration of secreted Shh has 0.7ng/ml in differentiation media and 0.85 ng/ml in conditioned media.
A. B.
Fig. 6. Preparation of 5E1, a neutralizing antibody against Shh. 5E1 reacted with secreted ShhN in ELISA at dilutions up to 1:1000~5:000 (ascitic fluid) (A). The titer of 5E1 was determined by ELISA using P19/hShhN conditioned medium as an antigen and its purified by affinity chromatography (B). Purified 5E1 were measured the concentration of ShhN by coomassie blue staining (C).
Mouse IgG5E1 dilution of 5E1 monoclonal antibody (mAb) or IgG and the total living cell number were measured by Trypan blue dye exclusion method.
4. Shh induces proliferation of P19 cells
To test whether Shh regulates P19 cell growth and differentiation, we cultured cells for 96hr in the presence of 5E1, as Shh neutralizing antibody, or control IgG and analyzed the cultures by cell counting using Trypan-blue dye exclusion method.
These assays were performed in the presence or absence of specific Shh inhibitor, the Shh neutralizing monoclonal antibody 5E1. Purified 5E1 (10µ g/ml) was added to the medium and the medium was replaced every 2 days. Cultures grown in P19 cells demonstrated low proliferation rate. However, a 2-fold, significant increase in cell proliferation was observed in P19/hShhN. P19/hShhN cells were almost completely inhibited by 5E1 monoclonal antibody, but unaffected by mIgG (Figure 8 A). The hShhN effect was blocked by 5E1. To test whether secreted Shh regulates proliferation of P19 cells, we cultured for 4 days in P19 cells conditioned medium and P19/hShhN cells conditioned medium. Conditioned medium from P19/hShhN increased the proliferation of P19 cells, which was blocked by 5E1 (Fig 8 B). Unlike P19 cells, the proliferating potential of P19/hshhN cells were maintained under the differentiation condition of P19 (Fig 8 C).
To confirm the effect of Shh on P19 cell proliferation, proliferation was studied by using 5E1 monoclonal antibody. Neutralizing activity of 5E1 monoclonal antibody was studied by incubating the antibody with 5E1 or IgG. The MTT assay was then performed after 92 hours incubation. As shown in Figure 9, P19/hShhN cells increased proliferation and inhibited by 5E1 (10 ㎍/ml) by compared to the control groups (Fig 9).
A. B.
Fig. 8. Shh induces proliferation of P19 cells. P19 andP19/hShhN cells were plated at a density of 3 x 103 cells per well of 48-well plate and cultured with DMEM supplemented 10% FBS (A), 0.5% FBS (B) and N2 (C) for 4 days. Cells incubated with 10µg/ml of 5E1 monoclonal antibody (mAb) or IgG and the total living cell number were measured by Trypan blue dye exclusion method. Data are expressed as a mean of duplicate per time point.
0 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. P19, P19/hShhN cells in suspension (500µl) were added to each well of a 4-well culture plate for a final concentration of 5x103 cells/well with 5E1 (10µg/ml) or IgG (10µg/ml) and incubated for 24h, 48h, 72h and 96h at 37°C in a CO2 incubator. At appropriate time points, 300µl of 0.5 mg/ml MTT solution were added to each well, and the cultures further incubated for 2h. Subsequently, the medium with MTT was replaced with 0.2 ml dimethyl sulphoxide. Sample (100µl) were added to each well of a 96-well culture plate. A microplate reader was used to measure absorbance at 570 nm for each well.
5. Shh induces neuronal differentiation of P19 cells
In vitro neuronal differentiation of P19 cells and P19/hShhN cells were grown without or with 10µM forskolin in the presence of N2 supplement for 4 days.
P19/hShhN cells formed neurite-like processes and showed a neuron-like morphology on 4 days (Figure 10 A). RT-PCR analyzed for differentiation of P19 and P19/hShhN cells into neural stem/precursors (Nestin), neurons (NF-M) (Fig 10 B). Expression of a neuron-specific protein, NF-M and β-tubulin III were increased in P19/hShhN, which were further increased by addition of 10µM forskolin.
Expression of proteins specific for oligodendrocyte (CNPase) and astrocytes (GFAP) were not obvious (Figure 10 C). Furthermore, immunocytochemistry revealed that cells were positive for neuron-specific markers Tuj1, suggesting that they could be induced into neurons (Figure 10 D).
A.
C.
Fig. 10. Neuronal differentiation of P19, P19/hShhN cells. Morphological changes of P19 cells by Shh or Foskolin (10µM) were examined under a phase contrast microscope (Magnification : x200) (A). Expression of Nestin, as neural stem cell marker, and NF-M, as neuronal marker, mRNA was measured by RT-PCR (B).
Neuronal differentiation was measured by western blot analysis with anti-Tuj1 and NF-M specific antibody (C) and Immunocytochemistry with anti-Tuj1 specific antibody (D).
6. Shh promotes neuronal progenitor cell/ motor neurons survival or proliferation
Nestin is expressed in the myotome the neural progenitor cells of the developing embryo. While the regulatory region for expression in myotome resides in the first intron of the Nestin gene, that for expression in neural progenitor cells exists in the second intron. To ascertain if Shh was important for neuronal progenitor cell survival we added Shh conditioned medium, as a secreted Shh, to cultures of P19/pNPEeGFP cell lines (Figure 11 A). By 4 days in culture, <4% of Nestin-positive cells were present in the control conditioned medium, whereas 25-30%
survive in Shh conditioned medium. GFP-positive cells were increased by secreted Shh (Figure 11 B). The expression of Nestin was further confirmed by RT-PCR. Shh increased the GFP and Nestin expression driven by the Nestin promoter/enhancer (Figure 11 C).
A.
Fig. 11. eGFP-labeled neuronal precursors in P19/pNPEeGFP. Construct for generation of P19/pNPEeGFP (A). P19/pNPEeGFP cells were cultured in Shh conditioned medium or control conditioned medium and counted GFP-positive cells (B). RT-PCR analyzed expression of Nestin (C).
7. Shh induces motor neuron differentiation in P19 cells.
The induction of MN progenitors depends on Shh activity (Briscoe and Ericson, 2001). To examine whether the spinal progenitors present in P19/hShhN can differentiate into MN progenitor, we monitored the expression of homeobox domain and bHLH transcription factors. In vitro neuronal differentiation of P19 cells was induced by Shh and forskolin. The P19/ShhN cells showed the morphology of the cells changed gradually. P19/hShhN cells formed neurite-like processes and showed a neuron-like morphology on 4 days. To examine whether the spinal progenitors present in P19/hShhN cells can differentiate into MN progenitors, we monitored the expression of HD and bHLH transcription factors that delineate sets of neural progenitor cells. Spinal MN progenitors are found in P19/hShhN cells and expressed Nkx6.1, Olig2 and Ngn2. To determine whether MNs are generated from progenitor cells present in P19/hShhN cells, we analyzed the expression of Hb9 and ChAT using RT-PCR and Immunocytochemistry. P19/ShhN cells increased expression of Hb9 and ChAT, suggesting that they could be induced into motor neurons (Fig 12).
Immunocytochemistry revealed that the differentiated P19/hShhN cells increased positive for motor neuron specific markers including Hb9 and ChAT (Fig 13 and 14).
0 1 2 3 4 1 2 3 4
0 µM FSK 10 µM FSK
Nkx6.1 Olig2 Ngn2 NeuroD
GAPDH ChAT Hb9
P19
Nkx6.1 Olig2 Ngn2 NeuroD
GAPDH ChAT Hb9
P19/hShhN
Fig. 12. RT-PCR of the expression of HD, bHLH, and motor neuron specific markers in P19 and P19/hShhN. Neuronal induction of P19 cells was performed as described in materials and methods. P19 and P19/hShhN were treated with 10µM forskolin for 4 days. The efficiency of MN differentiation were measured by expression level of motor neuron marker using RT-PCT. (
-
/+ indicates that forskolin treatment)Hb9Tuj1Hb9Tuj1Hb9Hoechst
P19 P19/hShhN
Tuj1
10µM Fsk 0µM Fsk
10µM Fsk 0µM Fsk
Fig. 13. Shh expression induces motor neuron differentiation of P19 cells.
Neuronal induction of P19 cells was performed as described in materials and methods. P19 and P19/hShhN were treated with 10µM forskolin for 4 days. Cells were visualized by Hoechst (blue) and immuno-stained with neuron specific antibody, Tuj1 (green) and motor neuron specific antibody, Hb9 (red). (Magnification : x200)
P19 P19/hShhN
Tuj1
10µM Fsk 0µM Fsk
10µM Fsk 0µM Fsk
ChATTuj1ChATTuj1ChATHoechst
Fig. 14. Shh expression induces motor neuron differentiation of P19 cells.
Neuronal induction of P19 cells was performed as described in materials and methods. P19 and P19/hShhN were treated with 10µM forskolin for 4 days. Cells were visualized by Hoechst (blue) and immuno-stained with neuron specific antibody, Tuj1 (green) and motor neuron specific antibody, ChAT (red). (Magnification : x200)
8. Shh induces dopaminergic neuron and motor neuron differentiation in P19 cells
Shh is expressed in ventral regions of the spinal cord, hindbrain, midbrain, and diencephalons. The specification of MN progenitor by Shh signaling is mediated through the patterned expression of homeodomain (HD) and basic helix-loop-helix (bHLH) transcription factors (Briscoe and Ericson, 2001). Dopaminergic neurons are induced at E9 (rat) by combinatorial action of Shh and FGF8, released by the floor plate and isthmus. To examine whether P19/hShhN can differentiate into MN progenitors, we monitored the bHLH transcription factors by RT-PCR. Olig2, a bHLH transcription factor, increased the expression in P19/hShhN cells. The induction of MN progenitors depends on Shh activity. To determine whether DAs are generated from P19 cells by Shh, we examined the expression of transcription factor and other DAs specific protein in differentiation. We showed that Shh increased the expression of TH, Nurr1 and Olig2 in P19 cells with foskolin (Fig 15). To determine whether MN progenitors generated from RA-exprosed P19/hShhN in current method, we analyzed the expression of Olig2, TH, and Nurr1 protein expressed selectively.
RA-exposed P19/hShhN increased expression of Olig2 and decreased expression of TH and Nurr1. In the Immunocytochemistry, P19/hShhN cells increased expression of Hb9 and TH, but no Hb9+ cells co-expressed TH, a marker dopaminergic neurons (Fig 16). These results provide evidence that Shh signaling is significant the induction of DAs and MNs in current differentiation method.
TH
P19 and P19/hShhN cells were differentiated for 4days with or without RA(0.5µM) in N2 media. RT-PCR analyzed expression of TH and Nurr1, as a Dopamine neurons marker and Olig2, as a Motor neurons transcription factor.
THHb9THHb9Hoechst THHb9THHb9Hoechst
P19 P19/hShhN
10µM Fsk 0µM Fsk
10µM Fsk 0µM Fsk
Fig. 16. Shh expression induces dopamine neurons and motor neurons differentiation of P19 cells. Neuronal induction of P19 cells was performed as described in materials and methods. P19 and P19/hShhN were treated with 10µM forskolin for 4 days. Cells were visualized by Hoechst (blue) and immuno-stained with dopamine neuron specific antibody, TH (green) and motor neuron specific antibody, Hb9 (red). (Magnification : x200)
9. Shh promotes neuronal differentiation in P19 cells without treatment of RA or aggregation culture.
Our previous study suggested that P19 cells, when replated as the aggregates, could differentiate into neural cells with RA (McBurney et al., 1987). However, P19/hShhN cells could differentiate into neurons without RA or aggregation culture.
To compare whether P19 cells were differentiated into neurons by Shh without treatment of RA or aggregation culture, P19 and P19/hShhN cells induced differentiation with and without treatment of RA or aggregation culture. Cells were differentiated in the absence or presence of 10µM forskolin. RT-PCR analysis of RNA isolated from differentiated P19 and P19/hShhN cells, which indicated that the expression of marker for neuron, NFM, for DAs, TH and Nurr1, and for MNs, Olig2 increased by Shh in current differentiation, but not in conventional differentiation (Figure 17). P19/hShhN cells could differentiate into neuronal cells without treatment of RA or aggregation culture. Compared to the conventional method, the current differentiation method without aggregation may provide a useful system for the study of the functions of Shh.
Nestin
Aggregate Agg. + Differentiation Nonagg. + Differentiation hShhN
Fig. 17. Shh promotes neuronal differentiation in P19 cells without treatment of RA or aggregation culture. P19 cells were differentiated into neurons by Shh without treatment of RA or aggregation culture, P19 and P19/hShhN cells induced differentiation with and without treatment of RA or aggregation culture. Cells were differentiated in the absence or presence of 10µM forskolin. RT-PCR analyzed expression of TH and Nurr1, as a Dopamine neurons marker and Olig2, as a Motor neurons transcription factor.
IV. DISCUSSION
In this study, we have further addressed the function of Shh in the proliferation and the motor neuron differentiation of P19 cells. Our studies reveal that overexpression of Shh can direct motor neuron differentiation of P19 cells in the absence of RA, when P19/hShhN cells were replated as the single sell suspension.
Furthermore, we show that Shh-overexpressing P19 cells differentiate exclusively into motor neuron.
Chemical inducers and aggregation are two key elements to impart fate choices of P19 cells. With aggregation, DMSO directs P19 cells to differentiate into mesoderm origin muscle cells. RA, however, induces P19 cells to differentiate into ectoderm-derived neural cells including neurons and astrocytes (E.M. Jones-Villeneuve et al., 1982, 1983; M.W. McBurney et al., 1982). According to the treatment of cells, the neural differentiation of RA-induced P19 cells can be divided into two sequential stages, a stage of induction and a stage of differentiation. During the first stage, P19 cells are allowed to aggregate in the Petri dish and induced with RA for four days. Based on cell morphology changes and gene expression profiles, it seems that pluripotent P19 embryonic carcinoma (EC) cells are determined into neural progenitor cells during the first RA-induction stage. In second stage, the induced P19 cells are replated into cell culture dish as the single cell suspension or aggregates and left to differentiate into mature neurons and astrocytes.
Shh could induce proliferation by a number of intracellular mechanisms.
Binding of Shh to Ptc releases this receptor’s repression of Smo, which then transduces the signal by acting on the transcription factor of Gli family. The transcriptionally activating Gli forms then upregulate Shh targets, including ptc, gli1, gli2 and Shh itself. Shh signaling may directly regulate the cell cycle, as it can
upregulate the expression of G1-phase cyclins of type D and E, and Pathed can act directly on the G2-phase cyclin B. Alternatively, Shh other activity may activate other signaling system that control cell proliferation. We observed that the Shh receptor Pathed and Smoothend and the target gene Gli is expressed in P19 cells. We have showed that Shh regulates the proliferation of P19 cells using growth kinetics and MTT assay. P19/hShhN cells increased the number of total cells in growth and differentiation condition and neutralized by 5E1. The biological activity of the Shh conditioned media was almost completely inhibited by 5E1, but unaffected by IgG.
The results suggest that the secreted hShhN was responsible for the stimulation of P19 cell proliferation .We conclude that Shh directly promotes P19 cells proliferation in vitro.
As the neurogenesis in vivo, neurons appear earler than glial cells during RA-induced P19 cell neural differentiation (E.M. Jones-Villeneuve et al., 1982).
Interestingly, our results show that P19/hShhN cells differentiate restrictively into neurons, but not glial cells. Our previous study suggested that P19/ShhN, when replated as the aggregates, could differentiate into neural cells without RA induction.
However, RA-inducted P19 cells were normally replated as the single cell suspension to enrich the cells of interest during their neural differentiation (M.W McBurney et
al., 1987, 1995). The wild-type P19 cells were used as the negative control. At day 3, P19/hShhN cells started to send out long neurite-like process. At day 4, P19/hShhN cells sent out many neurite-like process to form the networks. In parallel, the expression of Tuj1 and NF-M, as a neuronal specific marker, increased during the differentiation of P19/hShhN. This suggests that Shh may promote the neuronal differentiation in P19 cells without RA or aggregation culture.
Shh is necessary for the induction of both spinal motor neurons and midbrain dopaminergic neurons. In the developing midbrain, Shh was first characterized for its
Shh is necessary for the induction of both spinal motor neurons and midbrain dopaminergic neurons. In the developing midbrain, Shh was first characterized for its